Effect of TDZ treatment on the regeneration of shoots in recalcitrant grain legumes: Cicer arietinum, Glycine max, Phaseolus vulgaris, and Vigna unguiculata

In vitro regeneration remains a major bottleneck for the genetic improvement of recalcitrant grain legumes. The present study evaluated a TDZ-mediated flooding approach for rapid and high-frequency apparent direct shoot regeneration, with no visible callus formation based on gross morphological observations, in Cicer arietinum (black gram and white gram), Glycine max (soybean), Phaseolus vulgaris (common bean), and Vigna unguiculata (cowpea). Seedlings raised on N 6 medium containing 1.0 µM TDZ produced 4.5 shoots/seedling in black gram, 2.45 in white gram and 1.9 in common bean, while soybean and cowpea showed no shoot regeneration. Flooding TDZ-raised seedlings with distilled water and sucrose (D.W + S) enhanced shoot formation, whereas flooding with N 6 medium supplemented with 1.0 µM TDZ produced the strongest response, yielding 17.7, 16.4, 24, 23 and 41 shoots/seedling in black gram, white gram, soybean, common bean and cowpea, respectively. The maximum regeneration was recorded in cowpea (41 shoots/seedling), followed by soybean (24), common bean (23), black gram (17.7) and white gram (16.4). The regeneration response was associated with shoot-apex expansion and formation of additional shoot-forming regions, with no visible callus observed. These findings demonstrate that TDZ flooding substantially enhances shoot regeneration and extends its application to additional recalcitrant grain legumes. These findings demonstrate that the timing and mode of TDZ delivery are critical determinants of regeneration efficiency and extend the flooding approach to additional recalcitrant grain legumes. The protocol provides a simple, regeneration platform with potential applications in genetic transformation and genome editing.